Alexa fluor 488 goat anti rabbit igg

The paraffin sections were dewaxed using xylene for 15 min three times and hydrated in 100%, 90%, 80%, and 70% EtOH. Antigen retrieval was performed by a microwave in boiling antigen retrieval solution (pH 6.0, Dako, CA, USA) for 2 min 20 s. The sections were stained with the rabbit Ki67 antibody (Abcam, IA, USA, 1:300) overnight at 4 °C, and then incubated with secondary antibodies, such as Alexa Fluor 594 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, NY, USA, 1:1000), for 1 h at room temperature with 4,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). For cell staining with phospho-EGFR1, phospho-ErBb4, or phospho-Hck antibodies, the cells were fixed with 4% paraformaldehyde for 30 min at room temperature; washed with PBS; and incubated with phospho-EGFR1 (Abcam, 1:300), phospho-ErBb4 (Abcam, 1:300), or phospho-Hck (Abcam, 1:300) antibodies overnight at 4 °C. The samples were then incubated with Alexa Fluor 594 goat anti-rabbit IgG or Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, NY, USA, 1:1000) secondary antibodies for 1 h at room temperature with DAPI. Images of immunofluorescence staining were captured using a ZEISS LSM700 confocal microscope.

Immunohistochemical analysis of HDV antigens (HDAg) was performed using rabbit anti-HDAg antiserum as previously characterized [27 (link), 28 (link)] for human salivary gland tissues. Staining of formalin fixed paraffin embedded (FFPE) human salivary gland tissue was performed using citrate (10mM sodium citrate, pH6.0, 0.05% Tween20) antigen retrieval, followed by 30 minute incubation in blocking solution (3% BSA in TRIS buffered saline [TBS]) at room temperature, incubation with primary antibody overnight at 4°C (1:400 for rabbit antiserum) and 1 hour room temperature incubation in labeled secondary antibody solution (Alexa Fluor 488 Goat Anti-Rabbit IgG, 1:500, Life Technologies). Slides were then mounted in Fluoromount G (Electron Microscopy Systems) containing DAPI counterstain. B-cell, (B220, Abcam catalog #AB64100, 1:250) and T-cells (CD3, Abcam, catalog #AB16669, 1:100) were detected in FFPE mouse tissues using the manufacturer's recommended conditions. Secondary species-specific antibodies for B-cell b220 (Alexa Fluor 594 Goat Anti-Rat IgG, 1:500, Life Technologies) and T-cell CD3 (Alexa Fluor 488 Goat Anti-Rabbit IgG, 1:500, Life Technologies) were incubated for 1 hour at room temperature and mounted in Fluoromount G (Electron Microscopy Systems) containing DAPI counterstain.

Isolated hepatocytes or LSECs were cultured on a coverslip (AGC Techno Glass Co., Shizuoka, Japan), fixed with paraformaldehyde for 5 min and blocked with SuperBlock blocking buffer (Thermo Fisher Scientific). The cells were incubated with an E-cadherin antibody (BD Biosciences, San Jose, CA, USA) and VE-cadherin antibody (Abcam) at 1:200 for 1 h at room temperature. Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) were used as secondary antibodies for 1 h at room temperature with protection from the light. The cells were counterstained with DAPI and analyzed by fluorescence microscopy (Keyence Corporation, Osaka, Japan).
Mouse livers were mounted in Tissue-Tek O.C.T. compound (Sakura Finetek Japan Co Ltd., Tokyo, Japan) and frozen until preparation. Frozen sections were cut at 10-μm thickness and fixed in methanol for 10 min. Blocking was performed in PBS with 3 % bovine serum albumin (BSA). The slides were incubated with BMPER antibody (Abcam) at 1:200 in PBS overnight at 4 °C, and subsequently incubated with CD31 antibody (BD Pharmingen) at 1:100 in PBS for 1 h at room temperature. Alexa Fluor 594 goat anti-mouse IgG and Alexa Fluor 488 goat anti-rabbit IgG (Life Technologies) were incubated at 1:200 in PBS for 1 h at room temperature. The slides were counterstained with DAPI and analyzed using fluorescence microscopy.

Tissues were fixed upon slides with acetone, and then permeabilized with 0.1 µg/ml Triton X-100 in PBS. Tissues were incubated in 2.5% normal horse serum (Vector, Burlingame, CA, USA), followed by treatment with rabbit anti-ET-B antibody (Abcam Inc., Cambridge, MA, USA; 1:4000 dilution) and mouse anti-PMEL17 antibody (DAKO; 1:40). Tissues were incubated with Alexa Fluor^® 488 goat anti-rabbit IgG and Alexa Fluor^® 594 goat anti-mouse IgG (both from Life Technologies; 1:500), followed by nuclear staining with 4′6-diamidino-2-phenylindole (DAPI) (Life Technologies; 1:2000). Slides were mounted in Fluoromount-G^® (Southern Biotech, Birmingham, AL, USA). Co-cultured NHEKs and NHEMs in 4-well culture slides were fixed with 4% paraformaldehyde in PBS and were quenched by 50 mM NH₄Cl in PBS. The cells were then permeabilized with 50 µg/ml digitonin in PBS and were then incubated in PBS containing 0.2% gelatin, followed by treatment with rabbit anti-p62 antibody (MBL; 1:500) and mouse anti-PMEL17 antibody (DAKO Inc.; 1:500). Cells were then incubated with Alexa Fluor^® 488 goat anti-rabbit IgG and Alexa Fluor^® 594 goat anti-mouse IgG (both from Life Technologies; 1:1000). Slides were mounted in ProLong^® Gold Antifade Reagent with DAPI (Life Technologies), and images were obtained with a Leica DM5500B digital microscope (Leica Microsystems, Bannockburn, IL, USA).